MECHANISTIC STUDIES OF PMA AND FLAVOPIRIDOL INTERACTIONS

该项目的目标是为白血病治疗中结合分化诱导剂（如PMA、苔藓虫素、丁酸钠等）和药物周期蛋白依赖性激酶抑制剂（如黄匹吡醇、FP）的新治疗策略确定一个机制基础。最近的证据表明，在白血病细胞中，PMA和FP联合使用可显著诱导细胞凋亡，显著降低克隆原性，阻断PMA相关的成熟，并失调CDKI p21（WAF1/CIP1）的表达。我们假设这些行为源于fp介导的pma诱导的分化和/或相关的G，阻滞程序的中断。具体来说，我们提出FP通过1)阻断p21（WAF1/CIP1）的表达（一种凋亡抑制剂）来干扰pma相关的成熟；2)加速pRB去磷酸化，从而失调正常G，阻止事件；3)破坏E2F-1/pRB结合，从而促进e2f1相关基因的不适当表达，从而引发细胞凋亡。这些假设将在各种U937转染和亲代白血病细胞系中进行测试。还将寻找FP与其他分化诱导剂（如丁酸钠和苔藓抑素）之间协同作用的证据，并确定这些组合的共同特征。最后，我们将使用序列cDNA微阵列分析来鉴定凋亡、分化和细胞周期相关基因的谱，这些基因的表达可能被PMA/FP共处理所干扰。我们的最终目标是为结合分化诱导剂和CDK抑制剂提供一个合理的基础，最终可能转化为AML和其他恶性肿瘤的新治疗方案。

The goal of the project is to define a mechanistic basis for a novel therapeutic strategy combining differentiation-inducing agents (e.g., PMA, bryostatin, sodium butyrate, etc.) with pharmacologic cyclin-- dependent kinase inhibitors (e.g., flavopiridol; FP) in leukemia treatment. Recent evidence indicates that co-administration of PMA and FP in leukemic cells dramatically induces apoptosis, markedly reduces clonogenicity, blocks PMA-related maturation, and dysregulates expression of the CDKI p21(WAF1/CIP1). We hypothesize that these actions stem from FP-mediated disruptions in PMA-induced differentiation and/or the associated G, arrest program. Specfically, we propose that FP interferes with PMA-related maturation by 1) blocking the expression of p21(WAF1/CIP1), an inhibitor of apoptosis; 2) accelerating pRB dephosphorylation, thereby dysregulating normal G, arrest events, and 3) disrupting E2F-1/pRB binding, thereby promoting the inappropriate expression of E2F1-related genes that trigger apoptosis. These hypotheses will be tested in a variety of U937 transfectant and parental leukemia cell lines. Evidence of synergism between FP and other differentiation-inducing agents (e.g. sodium butyrate and bryostatin) will also be sought, and features common to these combinations identified. Finally, we will use serial cDNA microarray analysis to identify a spectrum of apoptosis -, differentiation-, and cell cycle-related genes whose expression may be perturbed by PMA/FP co-- treatment. Our ultimate goal is to provide a rational basis for combining differentiation-inducers and CDK inhibitors that may eventually be translated into novel treatment protocols for AML and other malignancies.